Stromal stem cells

ABSTRACT

Stromal stem cells are prospectively isolated from human bone marrow then expanded into clonal populations and cultured and used, the isolation being on the basis of expression of a cell surface marker, wherein the cell surface marker binds an antibody and wherein said antibody cross reacts with a cell surface marker found on mouse stromal stem cells or rat stromal stem cells, and optionally also on a cell of at least one other mammalian species selected from mouse, rat, horse, rabbit and pig cells. Useful stromal stem cell populations are positive for SDC2.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/470,492, filed Sep. 9, 2021, which is a continuation of U.S.application Ser. No. 16/009,048, filed Jun. 14, 2018, issued as U.S.Pat. No. 11,142,747 on Oct. 12, 2021, which is a divisional of U.S.application Ser. No. 15/089,435 filed Apr. 1, 2016, issued as U.S. Pat.No. 10,907,131 on Feb. 2, 2021, which is a divisional of U.S.application Ser. No. 14/377,597, a Section 371 U.S. national stage entryof International Patent Application No. PCT/EP2013/052692, internationalfiling date Feb. 11, 2013, issued as U.S. Pat. No. 11,230,700 on Jan.25, 2022, which claims priority to GB1202319.8, filed Feb. 10, 2012.

INTRODUCTION

The present invention relates to methods of isolating stem cells, tostem cell populations obtained from the isolated cells and to uses ofthose populations and of cells and tissue derived therefrom.

BACKGROUND

In the 1960s-70s, Friedenstein and colleagues demonstrated thatosteogenic potential—revealed by heterotopic transplantation of bonemarrow (BM) cells—was associated with a minor subpopulation ofBM-mononuclear cells (MNCs) (reviewed in Friedenstein, 1990). These MNCswere distinguishable from the majority of hematopoietic MNC by theirrapid adherence to plastic tissue culture vessels and by thefibroblast-like appearance of their progeny in culture, suggesting anorigin from the stromal compartment of BM. As well as establishing BMstroma as the source, Friedenstein, Owen and colleagues provided asecond breakthrough by showing that seeding of BM cell suspensions atclonal density results in the establishment of discrete coloniesinitiated by single cells (colony-forming unit fibroblastic, CFU-F[Friedenstein et al., 1970]).

Friedenstein and Owen later called this CFU-F generating cell theStromal Stem Cell (SSC) (Owen and Friedenstein, 1988) and references toSSC herein are based on that original cell definition.

BM-derived SSCs can be identified in a mixed population ofplastic-adherent (PA), fibroblastic, MNCs that give rise to bone, fat orcartilage and secrete potent immunomodulatory and angiogenic proteins.Preclinical studies demonstrate that PA-SSC mediate potentimmunomodulatory and angiopoietic responses in vivo. Currently, clinicaltrials are testing PA-SSC in 40 distinct degenerative, autoimmune andischemic diseases.

In the human marrow, approximately 1 BM mononuclear cell (MNC) in every80,000 MNC is a CFU-F forming SSC. To date, the most simple andfrequently used method of isolating these SSC from BM is dependent uponthe previously noted adherence to tissue culture plastic, according towhich the MNC are left to incubate for 10-14 days and in the interimCFU-F will attach and form colonies at a recognised frequency of1:80000. At 10-14 days these CFU-F are harvested by trypsin digest andreplated in serum-rich media at a density of 3-8000 CFU-F per cm². TheseCFU-F are then propagated in vitro until sufficient cell numbers areobtained to permit biochemical and cytological assessment. This approachis widely used but is regarded as inadequate for defining or purifyingSSC for clinical use, as only 1:80,000 BMMNC plated are SSC and themethods will not comply with good manufacturing protocols needed forclinical approval of related products.

Hence, in the prior art, stem cell populations have been identifiedbased on an initial ability to adhere to a plastic surface. From thisinitial screen, cell populations are obtained as clonal populations fromindividual colony forming units on the surface. These have also beenlabelled in the literature “mesenchymal stem cells” though the term maybe incorrect as non-mesenchymal stem cells may be included within theisolated cells. In a known isolation approach, these known cellpopulations are derived from stem cells that are positive for alkalinephosphatase (ALP) and CD271. Nevertheless, in clinical terms the cell isessentially unidentified.

Cell populations are prepared from these known isolated cells, such asby clonal expansion from a single, isolated cell, and used fortransplantation. The results are variable, however, in that thetransplanted cell populations sometime behave rather differently frombatch to batch, and with an element of unpredictability.

Prior art cell populations tend to form bone and fat and cartilage, butwith limited control, frequently making fat when bone or cartilage isrequired. Conversely, for instance where it is preferred to obtain cellsthat make fat, these fat-producing cells are only obtained unreliably.

A significant problem is that the starting cell population isessentially undefined, as isolation on the basis of adherence to plasticis not a sufficiently technical definition of a cell type. Even whenselected e.g. for the markers mentioned (ALP and CD271), expression ofthose markers in the cells or in progeny rapidly disappears uponculture, leaving an effectively undefined population. Useful propertiesof the cells also reduce or disappear over time—another problem with anundefined cell population.

OBJECTS OF THE INVENTION

It is an overall object of the invention to provide methods of isolationof stromal stem cells and cell populations and tissues derived therefromthat are at least an alternative to the art, and an object of particularembodiments of the invention is to provide methods that are improved,for example through increased definition of the cells obtained, or cellsand tissues that are improved, for example by increased reliability oftheir properties, rendering them more suitable for clinicalapplications.

SUMMARY OF THE INVENTION

The present invention is based upon prospective isolation of stromalstem cells, especially human stromal stem cells, based on expression ofmarkers or antigens that are expressed in a plurality of mammalianspecies. In methods and cell populations of the invention, cells aresorted on the basis of expression of a particular cross-species marker,this being referred to as prospective isolation, and then culture of thecells obtained, leaving to identification of cells, namely colonyforming units of fibroblasts (CFU-Fs), which can be clonally expanded.The cell population obtained after the clonal expansion is then proposedfor therapeutic, transplant and other uses.

Accordingly, the invention provides a method of isolation of a stromalstem cell, comprising isolation of a cell from a mixed population ofmammalian cells based on expression of a marker, wherein

-   -   the marker binds an antibody, and wherein    -   said antibody cross reacts with a marker found on a cell of at        least one other mammalian species selected from human, mouse,        rat, horse, rabbit and pig cells.

The invention provides a method of isolation of a stromal stem cell,comprising isolation of a cell from a mixed population of mammaliancells based on expression of the marker, wherein the marker is expressedby the mammalian cell and wherein a corresponding marker is alsoexpressed on a cell of at least one other mammalian species selectedfrom human, mouse, rat, horse, rabbit and pig cells.

Preferably the marker is found on human stromal stem cells and mousestromal stem cells, or on human stromal stem cells and rat stromal stemcells, or on all three. In embodiments, the marker is SDC2.

More specifically, the invention provides a method of isolation of ahuman stromal stem cell, comprising isolation of a cell from human bonemarrow that is negative for CD45 and positive for a further cell surfacemarker, wherein the further cell surface marker binds an antibody andwherein said antibody cross reacts with a cell surface marker found on acell of at least one other mammalian species selected from mouse, rat,horse, rabbit and pig cells. More specifically, the invention provides amethod of isolation of a human stromal stem cell, comprising isolationof a cell from human bone marrow that is positive for FAP alpha andpositive for a further cell surface marker, wherein the further cellsurface marker binds an antibody and wherein said antibody cross reactswith a cell surface marker found on a cell of at least one othermammalian species selected from mouse, rat, horse, rabbit and pig cells.

Preferably the further marker is found on human stromal stem cells andmouse stromal stem cells, or on human stromal stem cells and rat stromalstem cells, or on all three. In embodiments the further marker is NG2or, in particular, SDC2.

Methods of obtaining a population of cells are also provided, comprisingisolating cells according to the invention, and deriving the populationfrom those isolated cells; and methods of obtaining a clonal populationof cells are provided, comprising isolating a single cell according tothe invention and deriving a clonal population of cells from the singlecell.

The cells thereby obtained are also provided by the invention—hence,populations of stromal cells, preferably stromal stem cells, enrichedwith respect to the marker. Enrichment may be to 30% or more, 35% ormore, or 40% or more cells being positive for the marker. Inembodiments, the marker is NG2 or, in particular SDC2.

A specific population of cells of the invention is obtained by:—

-   -   providing a human stromal stem cell,    -   deriving a clonal population of cells from the human stromal        stem cell, and    -   optionally, further growing and/or expanding and/or passaging        the cells in culture, wherein the human stromal stem cell is        isolated from bone marrow, is negative for expression of CD45        and is positive for expression of a further cell surface marker,        wherein the further cell surface marker binds an antibody and        wherein said antibody cross reacts with a cell surface marker        found on a cell of at least one other mammalian species selected        from mouse, rat, horse, rabbit and pig cells.

A further specific population of cells of the invention is obtained by:—

-   -   providing a human stromal stem cell,    -   deriving a clonal population of cells from the human stromal        stem cell, and    -   optionally, further growing and/or expanding and/or passaging        the cells in culture, wherein the human stromal stem cell is        isolated from bone marrow, is positive for expression of FAP        alpha and is positive for expression of a further cell surface        marker, wherein the further cell surface marker binds an        antibody and wherein said antibody cross reacts with a cell        surface marker found on a cell of at least one other mammalian        species selected from mouse, rat, horse, rabbit and pig cells.

Products from the cells, such as bone, cartilage tendon and otherstromal stem cell products are provided by the invention, as are use ofthe cells e.g. in assays.

The invention enables identification of desired stromal stem cells onthe basis of specific marker expression, providing prospectivelypurified and defined cells and populations derived therefrom.

DETAILS OF THE INVENTION

Hence, the invention provides a method of isolation of a stromal stemcell, comprising isolation of a cell from a mixed population ofmammalian cells based on expression of a cell surface marker, wherein

-   -   the cell surface marker binds to an antibody, and wherein    -   said antibody cross reacts with a cell surface marker found on a        cell of at least one other mammalian species selected from        human, mouse, rat, horse, rabbit and pig cells.

The antibody may recognize a cell surface marker on a human cell andcross react with a cell surface marker on at least one other mammaliancell selected from mouse, rat, horse, rabbit and pig cells. In use, suchmethods are suitable for isolation of human, mouse, rat, horse, rabbitand pig cells, in particular human cells.

The antibody may recognize a cell surface marker on an equine cell andcross react with a cell surface marker on at least one other mammaliancell selected from human, mouse, rat, rabbit and pig cells. In use, suchmethods are suitable for isolation of human, mouse, rat, horse, rabbitand pig cells, in particular equine cells.

Similarly, the invention provides a method of isolation of a stromalstem cell, comprising isolation of a cell from a mixed population ofmammalian cells based on expression of a cell surface marker, whereinthe cell surface marker is expressed by the mammalian cell and wherein acorresponding cell surface marker is also expressed on a cell of atleast one other mammalian species selected from human, mouse, rat,horse, rabbit and pig cells.

The marker may be expressed on a human cell and the corresponding markermay be expressed on at least one other mammalian cell selected frommouse, rat, horse, rabbit, and pig cells, for isolation e.g. of humancells. The antibody may be expressed on an equine cell and thecorresponding marker expressed on at least one other mammalian cellselected from human, mouse, rat, rabbit and pig cells, for isolatione.g. of equine cells. A marker is a corresponding marker if an antibodycan be used to sort human cells based on binding to a marker on humancells and that same antibody can be used to sort cells of anothermammalian species.

Prospective stromal stem cell isolation of the invention thus usesmarkers found across species, referring to binding of markers indifferent mammalian species to a common antibody. In a specificembodiment described below, antibody to human SDC2 binds to and can beused to isolate human stromal stem cells and binds also to and can beused to isolate stromal stem cells in mouse, rat, horse and rabbit.

The antibody may bind to or cross react with markers on cells of atleast three mammalian species, at least four or at least five species.

Separately, the invention provides a method of obtaining or deriving astromal stem cell population comprising prospective isolation of cellsbased on expression of a marker that is similarly expressed in human andat least one other of mouse, rat, rabbit, horse and pig cells, forisolation e.g. of human stromal stem cells.

In embodiments of-the invention, stromal stem cells are isolatedaccording to expression of a marker expressed in at least human andmouse stromal stem cells, in at least human and rat stromal stem cellsor in at least human, mouse and rat stromal stem cells. Hence, forexample, an antibody to SDC2 can be used to isolate correspondingpopulations of stromal stem cells in human, mouse, rabbit, horse andrat. Other known antibodies, to NG2 and FAP alpha, details of which arebelow, can be used to isolate corresponding populations of stromal stemcells in human, mouse and rat. Corresponding stromal stem cellpopulations, and derivatives thereof, from both or all three species cantherefore be obtained in parallel or for comparison or to performanalysis of stromal cells of one species prior to work on stromal stemcells of another.

Various sources of the starting cells from which to isolate the stemcells are suitable. A source, a mixed population of mammalian cells, canbe bone marrow, adipose tissue, skeletal muscle, endometrium, placenta,umbilical cord, umbilical cord blood and Wharton's jelly. Sources ofhuman, mouse, rat, rabbit, equine and pig cells can be used, and inspecific examples human cells have been used. One source used inexamples is bone marrow, and a specific preferred source is human bonemarrow. Another source is cells, e.g. SSCs, derived from humanpluripotent cells. In an example, human SSCs from hES cells were used.

In use of the invention, an initial sort may be performed on the basisonly of the first marker, e.g. SDC2 expression. This method generallyalso isolates unwanted cells, meaning cells that are not stromal stemcells and may be e.g. B-cells or T-cells. By the further step ofisolating CFUs, however, these unwanted cells may be lost as they do notform colonies, so this level of sorting may be acceptable as only thedesired, stromal cells capable of forming CFUs will then produce clonalpopulations.

In certain embodiments of the invention, cells are isolated based onexpression of two separate markers. The combination selection of cellsmay be selection for positive/positive cells, meaning cells are selectedif they are positive for expression of the first marker and positive forthe expression of the second marker. The selection may also be forpositive/negative, for negative/positive or for negative/negative.

A combination method of the invention, typically combining one or moreor all features of any method described elsewhere heroin, thereforecomprises isolating cells on the basis of expression of a further cellsurface marker different from the first. This may be referred to as thesecond marker, though the nomenclature is to indicate merely that themarkers are different from one another and does not indicate a temporaldifference in the timing of selection or isolation according to thatmarker. The selection based on markers may be sequential, in eitherorder, though commonly is carried out in a single sorting or isolation,which can be simultaneous, as technology available enables this.

One suitable second marker is CD45. Suitably, cells that are CD45negative are selected. A further suitable marker is FAP alpha; suitablythe FAP alpha positive cells are selected. Suitable first cell surfacemarkers include SDC2 and NG2.

In another use of the invention, an initial sort is carried out on thebasis of CD45 expression, the negative fraction being selected. Aseparate sort is carried out on the basis of the first marker, e.g.SDC2, expression. The positive fraction can be taken or the negativefraction can be taken. In practice, the sorting is generally carried outsimultaneously. Cell viability can be dramatically reduced in sequentialsorting.

In a particular method of the invention, cells positive for the firstmarker are selected; these methods are suitable for isolation ofosteogenic cells and angiopoietic cells.

In another particular method of the invention, cells that are negativefor the first marker are selected; these methods are suitable forisolation of adipogenic cells.

A specific method of isolation of a human stromal stem cell comprisesisolation of a cell from human bone marrow that is positive for a firstmarker and negative for CD45, wherein the first marker binds an antibodyand wherein said antibody cross reacts with a marker found on a cell ofat least one other mammalian species selected from mouse, rat, horse,rabbit and pig cells. The first marker is preferably SDC2 and isolationuses an antibody that binds SDC2 and cross reacts with a correspondingmarker on all of human, mouse, rat, horse, rabbit. The first marker maybe NG2 and isolation uses an antibody that binds human NG2 and crossreacts with a marker corresponding to NG2 on one or more of mouse, rat,horse, rabbit and pig cells.

Combination marker sorting can also comprise sorting according to threedifferent markers, for example on the basis of the second marker andthen two or more first markers. Sorting by NG2 can be used to subdividethe SDC2 +ve population.

Specific isolated stromal stem cells of the invention are:—

-   -   (i) CD45 −ve, SDC2 +ve    -   (ii) CD45 −ve, SDC2 −ve    -   (iii) CD45 −ve, SDC2 +ve, NG2 +ve    -   (iv) CD45 −ve, SDC2 +ve, NG2 −ve    -   (v) FAP alpha +ve, SDC2 +ve    -   (vi) FAP alpha +ve, SDC2 −ve    -   (vii) FAP alpha +ve, SDC2 +ve, NG2 +ve    -   (viii) FAP alpha +ve, SDC2 +ve, NG2 −ve

From cells that have been isolated, cell cultures and populations can beobtained. This can be achieved by clonal expansion of an isolated cell(e.g. a cell that is at least initially CD45 negative and SDC2 positiveor CD45 negative and SDC2 negative) and then continued growth or cultureof the cells obtained. Note that the cells obtained by this continuedgrowth and culture and passaging tend initially to demonstrate the samemarker spectrum as the originally isolated cell or cells. Over time theexpression pattern may change. But the properties of the resultantpopulation are linked to the criteria of the initial isolation (e.g. acell that is at least initially CD45 negative and SDC2 positive or CD45negative and SDC2 negative).

From cells that have been isolated, cell cultures and populations cangenerally be obtained having a high homogeneity, measured by expressionof the marker or antigen used for the isolation. Hence, mammalianstromal cell populations are also provided by the invention expressinghigh levels of the first cell surface marker. The % of cells expressingthe first marker may be 50% or more, 60% or more, 70% or more, 75% ormore, 80% or more, or 90% or more. In a specific embodiment of theinvention described below in more detail, initial cell populationsexpress the first marker at a level of 95% or more. In referring tocells that are positive, reference is to positive as measured at or onthe cell surface, e.g. as detectable using a labelled antibody to themarker.

Cells populations may also be derived from the above, for example byculture and/or passaging, and in doing so the proportion of cells thatretain expression of the first marker may reduce, but neverthelessremain higher that in populations not selected on the basis of themarker. Hence, further mammalian cell populations are also provided bythe invention expressing high levels of the first marker. The % of cellsexpressing the first marker may be 30% or more, 40% or more, 50% ormore, or be at the levels recited immediately above. The cellpopulations of the invention have specified purity and are defined. Thecells can be identified/selected on the basis of marker expression andused immediately, with no need for culture to determine if asufficiently pure population of cells has been obtained.

In particular embodiments of the invention the marker is NG2 or,especially, SDC2.

A population of mammalian stromal stem cells is thus provided, wherein75% or more of the cells are positive for a cell surface marker, andwherein a corresponding cell surface marker is also expressed on a cellof at least one other mammalian species selected from human, mouse, rat,horse, rabbit and pig cells. The marker is suitably expressed on a humancell and the corresponding marker is expressed on a mouse cell andoptionally also on at least one other mammalian cell selected from rat,horse, rabbit and pig cells. The marker is suitably expressed on a humancell and the corresponding marker is expressed on a rat cell andoptionally also on at least one other mammalian cell selected frommouse, horse, rabbit and pig cells. In another exemplary population ofstromal stem cells, 75% or more of the cells are SDC2 positive, andthese cells were osteogenic. Further, the cells may additionally becharacterised by expression levels of the second marker. The % of cellsexpressing the second marker may be 50% or more, 60% or more, 70% ormore, 75% or more, 80% or more, or 90% or more. In a specific embodimentof the invention described below in more detail, 95% or more of cells inthe initial cell populations are positive for the second marker. Thecells may separately be negative for the second marker. The % of cellsnot expressing the second marker may be 50% or more, 60% or more, 70% ormore, 75% or more, 80% or more, or 90% or more. In a specific embodimentof the invention described below in more detail, 95% or more of cells inthe initial cell populations are negative for the second marker. In oneexemplary population of osteogenic and angiopoietic stromal stem cells,75% or more of the cells are SDC2 positive and 75% or more of the cellsare CD45 negative.

Cell populations are also provided by the invention expressing lowlevels of the first marker. The % of cells not expressing the firstmarker may be 50% or more, 60% or more, 70% or more, 75% or more, 80% ormore, or 90% or more. In a specific embodiment of the inventiondescribed below in more detail, initial cell populations express thefirst marker at a level of 5% or less or are regarded as negative forthat first marker. In one exemplary population of stromal stem cells,75% or more of the cells are SDC2 negative, and are adipogenic cells.Further, the cells may additionally be characterised by expressionlevels of the second marker. The % of cells expressing the second markermay be 50% or more, 60% or more, 70% or more, 75% or more, 80% or more,or 90% or more. The cells may separately be negative for the secondmarker. The % of cells not expressing the second marker may be 50% ormore, 60% or more, 70% or more, 75% or more, 80% or more, or 90% ormore. In a specific embodiment of the invention described below in moredetail, 95% or more of cells in the initial cell populations arenegative for the second marker. In one exemplary population ofadipogenic stromal stem cells, 75% or more of the cells are SDC2negative and 75% or more of the cells are CD45 negative.

Accordingly, the invention also provides a method of obtaining apopulation of cells, comprising isolating cells according to the methodsdescribed and deriving the population from those isolated cells; and theinvention further provides a method of obtaining a clonal population ofcells, comprising isolating a single cell according to the methodsdescribed and deriving a clonal population of cells from the singlecell. Generally, culture comprises obtaining an initial population ofcells and then further growing and/or expanding and/or passaging thecells in culture. SDC2 is the characterising marker in specific examplesdescribed below.

Cell populations described in specific examples below have been obtainedand found to exhibit useful properties. Hence, still further provided bythe invention are a population of cells obtainable according to thedescribed and claimed methods. The cell populations are preferablyhuman, equine, rabbit, mouse and/or rat cells.

A population of cells of a specific embodiment is obtained by:—

-   -   providing a human stromal stem cell,    -   deriving a clonal population of cells from the human stromal        stem cell, and    -   optionally, further growing and/or expanding and/or passaging        the cells in culture, wherein the human stromal stem cell is        isolated from bone marrow, is negative for expression of CD45        and is positive for expression of a further cell surface marker,        wherein the further cell surface marker binds an antibody and        wherein said antibody cross reacts with a cell surface marker        found on a cell of at least one other mammalian species selected        from mouse, rat, horse, rabbit and pig cells.

Tissues are provided by the invention, by obtaining cells according todescribed methods, and obtaining tissue therefrom. Tissue selected frombone, cartilage and tendon can be obtained in this way. Adipose tissueor tissue for reconstructive surgery can also be obtained.

A further use of the invention lies in providing cells for and assaysusing the isolated cells and progeny thereof. Hence, a method ofconducting an assay comprises obtaining cell according to the describedmethods, and using those cells in the assay.

Prior art cell populations, as mentioned, tended to form bone and fatand cartilage, with limited control and thus sometimes making fat whenbone or cartilage is required. Cell populations of the invention can beprepared that tend not to form fat. This is an advantage. Separate cellpopulations of the invention can be prepared that do indeed tend to formfat. The user may thus be provided with enhanced control andpredictability of the properties of the cells, based on a specific cellmarker selection criterion.

An advantage of specific cell populations of the invention can be putanother way, namely for the CD45 negative, SDC2 positive cells that theytend to be osteogenic, generating bone- and cartilage-producing cellpopulations with higher frequency. The prior art difficulty of mixedresults and low reproducibility of obtaining bone and cartilage maythereby be solved. Specific cell populations now obtainable may havefurther advantages; e.g. SDC2 positive populations appear to be moreangiopoeitic, inducing neighbouring cells to form vasculature. This isadvantageous in treating diseases that would benefit from improvedvasculature, for example ischemic diseases.

In addition, SDC2 positive populations are derived in specific methodsof the invention from starting cells that are more highly defined thanin the prior art, by reference to a marker that persists in cells andprogeny. In itself, this is an advantage. The cell population is anacceptably defined population.

A further potential advantage of the invention is that the marker usedfor prospective isolation of the initial cell population is also usefulfor prospective isolation of cell populations in other species. Thus, ina specific embodiment, SDC2 is common across all of human, mouse, rat,horse, rabbit. As a result, it is possible to isolate a correspondingpopulation in, say, mouse cells and then extrapolate from work on mousecells to work on human cells. A defined population in the mouse can beused to obtain data which can then be taken further, in a correspondingdefined population in another species, especially human cells. A problembelieved to be true for prior art mesenchymal stem cell populations,i.e. those obtained using a known prospective isolation, is that themarker used for isolation, say, of the mouse cells does not have acorresponding pattern of expression in the human cells. The inventionmay thus provide defined cell populations with cross species parallelpopulations, such as mouse and human or mouse and equine, etc. Thisfacilitate preclinical and clinical work because knowledge obtained fromexperiments carried on initially on cells of one species can betransferred to late work on the corresponding defined cell population inanother species.

Cells and tissue of the invention, and compositions comprising the cellsand tissues, can be used to treat various mammalian conditions anddiseases, including in particular those treatable using cells andproducts derived from existing SSC products. The cells and tissue mayinteract with dendritic cells and drive IFN-β secretion, and hence maybe used as a tumor suppressor. Cancers in general may be treated usingthe invention, specifically including hepatocellular carcinoma, cervicalcancer, pancreatic cancer, prostate cancer, fibrosarcoma,medullablastoma, and astrocytoma. Lung diseases may be treated includingAcute lung injury (ALI); Acute respiratory distress syndrome (ARDS);Chronic Obstructive Pulmonary Disorder (COPD); Idiopathic pulmonaryfibrosis (IPF). The cells and tissues may be used to treat sepsis andsepsis-induced multiorgan failure, bone marrow transplant (BMT) orhaematopoietic stem cell (HSC) rejection; solid organ transplant (SOT)rejection (including liver, kidney, skin, cornea, heart, lung); acutetoxin-induced liver failure; autoimmune hepatitis; primary biliarycirrhosis (PBC) and primary sclerosing cholangitis (PSC); osteonecrosis;degenerative disc disease; rheumatoid arthritis; osteoarthritis anddelayed bone healing in diabetic patients; autoimmune nephritisincluding Wegener's granulomatosis (WG); burns, severe burns; musclewasting conditions and atrophic syndromes including sarcopenia; cachexiaand other muscle wasting conditions including the muscular dystrophies(Duchenne and Becker); congestive heart failure, acute myocardialinfarction and stroke; type 1 diabetes; type 2 diabetes; diabeticretinopathy and other retinopathies; diabetic nephropathy and othernephropathies; diabetic neuropathy and other neuropathies; non-healingdiabetic ulcers; diabetic cardiomyopathy and other myopathies;athersclerosis; peripheral artery disease and critical limb ischemia;uveitis; (wet or dry) acute macular degeneration (AMD); retinal andcorneal damage; autoimmune conditions such as autoimmune gastritis(AIG); graft-versus-host disease (GvHD); multiple sclerosis anddemyelinating diseases; thyroid disease; inflammatory bowel diseasesincluding Crohn's Disease, Ulcerative colitis and fistulising Crohn'sdisease; scleroderma; lupus (SLE); Graves' Disease; and autoimmunelymphoproliferative disease (ALPS).

The cells and tissue may also be used to treat particular various equineconditions, including laminitis, tendon injuries and exercise inducedpulmonary haemorrhage (EIPH)—also known as “bleeding” or a “bleedingattack”.

Also provided by the present invention is a pharmaceutical compositionfor treating a disease or disorder in an animal, in particular a mammaland for example a human or horse. The pharmaceutical compositionsuitably comprises cells or tissue of the invention in an amounteffective to treat the disease or disorder in the animal. The cells maythus be administered with an acceptable pharmaceutical carrier. Forexample, the cells may be administered as a cell suspension in apharmaceutically acceptable liquid medium for injection. Examples ofliquid medium are saline, phosphate buffered saline, optionally alsocontaining additional materials such as dimethylsufoxide (DMSO) andhuman serum albumin. The cells and tissue may generally be administeredin a variety of formats as known for existing mesenchymal stem cell andlike products and tissue derived therefrom. They can be administeredsystemically, e.g. by intravenous infusion, or direct injection. Thecompositions may comprise a matrix or scaffold, or cells or tissue maybe administered by injection into a site already comprising matrix orscaffold in situ. The cells or tissue may thus be administered incombination with hyaluronic acid, collagen or other extracellularmatrix. Further formulation and administration examples that can beapplied mutatis mutandis to the cells and tissue of the invention may befound in the art, e.g. in WO2001080865, EP2545928 and WO1999061587. Amethod of treatment of an animal is provided, comprising administeringto the animal a composition of the invention. Cells or tissue accordingto the invention are provided for use in treatment of a disease ordisorder of an animal. Embodiments of the methods and uses compriseembodiments generally of the invention as described herein.

Suitable antibodies are available to the skilled person for performingsorting and isolation based on the identified markers. A human NG2/MCSPAntibody is available from R&D Systems, Inc. (614 McKinley Place NE,Minneapolis, MN 55413, USA), as a monoclonal Mouse lgG1 Clone #7.1,Catalog Number: MAB25851. A NG2 (G-20) antibody is available from: SantaCruz Biotechnology, Inc. (2145 Delaware Avenue, Santa Cruz, California,5060, USA), ref: sc-30923, reactive with mouse, rat, human, equine,canine, bovine and porcine; the blocking peptide, sc-30923 P, is alsoavailable. A Human Fibroblast Activation Protein a/FAP Antibody, catalogNumber: AF3715, is available from R&D Systems, Inc. A FAPalpha (Y-16)antibody is available from Santa Cruz Biotechnology, Inc. reactive withat least human, rat and mouse and also equine, canine, bovine, porcineand avian. An Anti-Fibroblast activation protein, alpha antibody,ab28243, is available from Abeam (330 Cambridge Science Park, Cambridge,CB4 0FL, UK) reactive with at least mouse, rat and human. A Syndecan 2antibody, orb13481, reactive with at least human, mouse and rat isavailable from Biorbyt Ltd. (12 Pembroke Avenue, Denny IndustrialCentre, Waterbeach, Cambridge, CB25 9QR, UK). The SDC2 antibody used inspecific examples, catalog number: MAB29651 (Clone 305507) is availablefrom R&D Systems, Inc, reactive with human, mouse, rat, equine, rabbitand pig.

SDC2, also called Fibroglycan and now, CD362, was originallybiochemically characterized as one of the major heparan sulfate (HS)glycosaminoglycan (GAG)-containing cell surface proteins expressed inthe lung. SDC2 is one of four members of this single-pass transmembranefamily in vertebrates. Herein, reference to “S2” and “SDC2” refer toSDC2.

The invention is now described in specific embodiments with reference tothe accompanying drawings in which:

FIG. 1 shows labelling by anti-SDC2 antibody of human stromal stem cellsbut not human lung fibroblasts. Green lines indicate anti-SDC2− APCstaining of cells. Red lines indicate labelling with appropriate controlantibody (Rat lgG2B Isotype Control-APC; R&D #IC013A; Clone −141945).Blue lines indicate positive control labelling of MRCS withanti-PDGFRa-APC antibody (R&D Systems #FAB1264A; Clone—PRa292);

FIG. 2 shows labelling by SDC2-APC antibody of CD271^(bright)/^(DC45low)human bone marrow mononuclear cells. Data show fluorescence-activatedCell Sorting (FACS) profile of 3.5 ×10⁷ BMMNCs stained withaforementioned SDC2-APC, CD271-PE and CD45-FITC (both from BD).CD45-FITC staining permitted gating of BMMNCs into 3 populations, (A)CD45-ve—BLUE, (B) CD45low—ORANGE, and (C) CD45high—GREEN. In (B) rare TPSDC+ve/CD271 bright/CD45low cells are BLUE;

FIG. 3 shows enhanced enrichment in CFU-F inSDC+/CD271^(bright)/DC45^(low) sorted bone marrow mononuclear cells.Data show fluorescence-activated Cell Sorting (FACS) profile 10⁷ BMMNCsstained with aforementioned SDC2-APC, CD271-PE and CD45-FITC (both fromBD);

FIG. 4 shows percentage of wells of a 96 well plate in which no cloneformed as function of number of SDC+/CD271^(bright)/CD45− mononuclearcells per cell;

FIG. 5 shows the number of population doublings of clones;

FIG. 6 shows in vitro GAG deposition of SDC2+ stromal stem cells at 2%and 19% oxygen tension. Representative Safranin-O stained histologysections from SDC2+SSC derived micromass pellets are shown;

FIG. 7 shows in vitro lipid deposition of SDC2+ and SDC2− (labelled asS2+ and S2− respectively) stromal stem cells;

FIG. 8 shows calcium deposition of SDC2+ and SDC2− stromal stem cells inresponse to in vitro osteogenic stimuli;

FIG. 9 shows GAG deposition of SDC2+ and SDC− stromal stem cells inresponse to in vitro chondrogenic stimulation;

FIG. 10 shows relative HUVEC cord formation of SDC2+ and SDC2− stromalstem cells in response to in vitro angiogenic stimulation;

FIG. 11 shows that rare CD45−/SDC+ human bone marrow mononuclear cellsexpress stromal stem cell marker CD271, CD146 and NG2;

FIG. 12 shows SDC2 expression of stromal stem cells isolated from andrabbit bone marrow;

FIG. 13 shows increasing levels of SDC2 in stromal stem cells derivedfrom equine bone marrow upon confluence, and stromal stem cells derivedfrom pig bone marrow express SDC2 in low oxygen tension;

FIG. 14 shows increasing levels of SDC2 in stromal stem cells derivedthree strains of rat bone marrow upon confluence;

FIG. 15 shows FACS isolation of CD45− mononuclear cell with co-stain forSDC2 and Scal; and

FIG. 16 shows enhanced enrichment in CFU-F in SDC+/Scal selected mousemononuclear cells.

We used a rat lgG2B monoclonal antibody to human SDC2 conjugated to theAllophycocyanin (APC) fluorochrome (R&D Systems number FAB2965A; clone305515) to indicate if expression of SDC2 protein is enriched on thecell surface of human SSC in comparison to a control human foetal lungfibroblast cell line, MRCS. MRCS lung fibroblasts cultured in SSC growthmedia (aMEM/10% PAA FSC; NUNC T175 flasks) did not express SDC2 (FIG. 1). As a control, we show that MRCS fibroblasts do express the PDGFRamarker (CD140a-APC). These data suggests that expression of SDC2 proteinis enriched on the surface of human SSC in comparison to control lungfibroblasts (FIG. 1 ).

At this time, the state of the art for antibody-based purification ofSSC from BM consists of using a combination of anti-CD271 (LNGFR) andanti-CD45 antibodies, reported by University of Leeds (Drs. McGonagleand Jones). This isolation of CD45low/CD271 bright cells has been shownto capture all CFU-F (SSC).

However, the definition of CD271 ‘bright’ cells can be difficult tostandardise from lab to lab. To investigate if this anti-SDC2 antibodyco-stains CD45low/CD271 bright BMMNCs, 30 ml of human BM was aspiratedfrom donor CRFG #0007 at the Clinical Research Facility (CRF) at GalwayUniversity Hospital (GUH) by Dr Ruth Morrell.

BMMNCs (5 ×10⁸) were isolated by Ficoll centrifugation, washed in PBS,resuspended in MACS buffer and blocked with Human FC-Block (Miltenyi,UK). BMMNCs (4 ×10⁷) were stained with anti-SDC2-APC (R&D),anti-CD271-PE (BD) anti-CD45-FITC (BD) and Sytox/DAPI viability dye.Cells were analysed by FACS on the Becton Dickinson Ariall at NUIGalway.

FIG. 2 indicates representative histogram/dot plots from SDC2/CD271/CD45triple stained cytometry experiments. FIG. 2B indicates BMMNCs thatexpress low/mid levels of the CD45 marker (orange). In agreement withother reports, we find that CD271-positive cells are found within theCD45low population (FIG. 2B) and in these experiments, we noted that theanti-SDC2-APC antibody labelled CD45low/CD271-positive cells.Specifically, the anti-SDC2-APC antibody labels CD45low/CD271 brightBMMNCs. The SDC2+/CD45low/CD271+ triple positive (TP) population arerare within BMMNCs with a frequency ranging from 1:16,000 to 1:23,000.

FIG. 3 shows that SDC2+/CD271+/CD45− MNC fraction contain 3000-fold moreCFU-F/S SC compared to native pre-sorted BMMNCs. Conversely theSDC2-negative fraction of the CD271+ population does not retain asignificant number of CFU-F/SSC

Single cell FACS sorting experiments were performed to enumerate thenumber of clonogenic cells within the SDC2+/CD271+ population. SingleSDC2+/CD271+/CD45− MNC were sorted at 1, 3 and 20 cells per well in a 96well plate. A limiting dilution analysis reveals that, at lcell perwell, 16-17 clones formed per 96 well plate (FIG. 4 ). All 16 cloneswere proliferative and able to undergo 15-20 population doublings (FIG.5 ). Notably, selected SDC2+ clones were able to undergo significantchondrogenesis in response to in vitro micromass culture. All fiveclones exhibited enhanced glycosaminoglycan (GAG) deposition whencultured in low (2%) tensions of oxygen (FIG. 6 ).

When compared to pre-sorted (parental) SSC, FACS-sorted and cultureexpanded SDC2+SSG exhibit significantly attenuated deposition of lipidsin response to in vitro stimulation with potent adipogenic media over a14 day differentiation regimen (FIG. 7 ), as visualised with Oil Red Ostaining, extractions and quantification. Conversely, compared toSDC2-SSC and pre-sort SSC, SDC2+ SSC elicit enhanced deposition ofcalcium and enhanced matrix mineralisation in response to a 14 dayinduction with an osteogenic media, as measured by calcium extractionand Alizarin Red S staining respectively (FIG. 8 ). Notably, nodifference was observed between the three populations of SSC whensubjected to chondrogenic micromass culture (FIG. 9 ).

Human vascular endothelial cells (HUVEC) can form angiogenic cord-liketubules within 24 hours of being plated on nutrient-rich matrigel.Co-culture of SDC2+ SSC at a ratio of 1:1 with HUVEC on matrigel elicitsa 3-fold increase in the number of stable vascular tubules at 24 hours(FIG. 10 ).

Finally, Human SDC2+/CD45− MNC also express key stromal markersincluding CD146, NG2 (CSPG4) and CD271 (FIG. 11 ).

FIG. 12 represents flow cytometry histograms of SSC derived from BM andAdipose MNC of goat and rabbit BM tissue. While the SDC2 marker does notappear to be detectable on goat SSC, significant levels of SDC2 proteincan be detected on rabbit SSC (FIG. 12 ).

An increased detection of SDC2 protein is increased in cultured equineSSC in response to confluent culture (FIG. 13 ). SDC2 protein can alsobe detected in a sub-population of porcine SSC when cultured in lowoxygen tension (FIG. 13 ).

SDC2 marker is expressed on the surface of rat SSC (FIG. 14 ). As seenin equine SSC, SDC2 protein increases in the surface of rat SSC inresponse to confluence. This pattern can be seen in SSC derived from themarrow of three typically used laboratory strains of rat, namely, DarkAgouti, Sprague Dawley and Lewis (FIG. 14 ).

EXAMPLES Example 1.1—Isolation of Bone Marrow Aspirates

Human bone marrow samples were obtained from the posterior iliac crestof healthy volunteers (n=3) following written consent from the patients.Patients underwent virology testing for HIV I and II, Hep A/C, HBsAg,Anti-HB core, Syphilis and CMV in accordance with EU Tissue Directive2006/17/EC requirements. In a BSC, samples are pooled and divided into7.5 mL aliquots and subjected to density-gradient centrifugation.

Example 1.2—Isolation and Expansion of Human SSC by Density-GradientCentrifugation (Ficoll)

In a biological safety cabinet under aseptic techniques, 7.5 mL ofFicoll is pipetted into 50 ml centrifuge tubes. To remove clots, the 30ml of BM was filtered through a 100 micron cell sieve (BD Falcon) into a50 ml centrifuge tube. Filtered marrow was diluted 1/1 in D-PBS and thensplit evenly between the 4 tubes containing Ficoll, slowly pipetting theBM onto the side of the tube lying at an angle of 35° to 45° to insure aslow release of BM, without disturbing Ficoll or producing bubbles.Tubes were then centrifuges for 22 mins at 900 g with centrifuge brakesset to zero to form a fractionated sample. After centrifugation, tubecontents formed three layers; a top layer of plasma, a thin layer—Buffycoat—contains the MNC, a clear layer of Ficoll and a bottom layercontaining red blood cells constituents—erythrocytes and granulocytes.The Buffy coat was carefully aspirated out being careful not to disturbthe surrounding cells. These cells were then transferred to another 50mL centrifuge tube and resuspended in 45 mls D-PBS. These tubes werethen centrifuged for 10 mins at 350 g. Supernatant was aspirated andpellets resuspended in 5 mL complete media. These were then centrifugedfor 10 mins at 350 g. Supernatant was aspirated and pellets were pooledin 5 mL D-PBS.

Example 1.3—CFU-F Plates Seeding

After isolation of mononuclear cells via direct plating or Ficoll, 9×10⁶ cells were isolated from both sets of cells and seeded in 10 cmdishes in triplicate at a seeding density of 3 ×10⁶ MNC/plate. Thesecells were washed and fed at same time as cells in culture as outlinedabove.

Example 1.4—Crystal Violet CFU-F Staining

On days 12-14, cells were fixed and stained for CFU-F analysis. Mediawas aspirated from plates and plates were washed three times in D-PBS toremove and remaining media. Cells were fixed by pipetting 8 mL 95%Methanol, stored at −20° C., onto cells for 10 mins and gently swirling.Methanol was aspirated from plates and cells were washed once withD-PBS. 8 mL of crystal violet (0.5% crystal violet, 99.5% Methanol) wasthen added to plates and plates were gently swirled. Plates were leftfor 10-15 mins. Excess crystal violet was aspirated and cells werewashed three times with D-PBS to remove and remaining excess stain.Plates were then inverted and left to dry overnight. Dry plates werethen imaged using a flat bed scanner. Colonies were then counted andcharacterised by visual inspection under an inverted light microscope(Olympus CKx41). Colonies comprised of clusters greater than 50+ werecounted as a CFU.

Example 2—Antibody Analysis of Mononuclear Cells and Stromal Stem Cells

Table 1 shows the details of the antibodies used to profile themononuclear cells and stromal stem cells produced in Example 1.

TABLE 1 Antibody Supplier Catalogue No. CD362/Sydecan-2 R&D Systems N/ACD271/LNGFR BD N/A W8B2/TNAP/ALP Miltenyi N/A TWEAK/TNFSF13 BD N/AAPRIL/CD256 BD N/A CD146 BD N/A CD105 Invitrogen MHCD10504 CD73 BD550257 CD90 BD 555596 CD14 AbD Serotec SPL2185 CD19 BD 345777 CD3 BD345765 CD34 BD 555822 CD45 BD 555483 ┌1, γ2a controls BD 342409 HLA-DRInvitrogon MHLDR04 ┌2b control Caltag MG2b04

Blocking Solution Preparation

Blocking solution was prepared by adding 1 mL of FBS to 49 mL of D-PBSin a 50 mL tube.

Sample Preparation

Cells were trypsinised at 37° C. and transferred to culture media in 15mL tube. Cells were centrifuged for 5 mins at 400 g. Supernatant wasaspirated and cells resuspended in 5 mL complete culture media. Cellcounts and viability testing were performed using Trypan blue. Cellswere then centrifuged for 5 mins at 400 g and supernatant aspirated.Blocking solution was then added to cell pellets to resuspend cells at 1×10⁶ cells/mL.

Staining of SSC (Analysis on FACS Canto)

PE-labelled antibodies were removed from refrigeration and placed on icealong with a 96 well round bottomed plate (Sarstedt) and blockingsolution. 1 ×10⁵ cells (100 μL) was pipetted into each of 12 wells ofthe 96 well plate on ice, one for each antibody and 1 for unstainedcells. Plate was then centrifuged for 4 mins at 400 g, 4° C. Supernatantwas aspirated carefully to not disturb cell pellet and 50 μL of blockingsolution was added to each well and pellet resuspended by pipetting ofsolution.

Example 3—Chondrogenic Differentiation of SSC

Table 2 shows the composition of the incomplete chondrogenic media(ICM).

TABLE 2 Reagent Volume Final Concentration DMEM (HG) 95 mL Dexamethasone1 mM 10 μL  100 nM Ascorbic acid 2-P: 5 mg/mL  1 mL   50 μg/mLL-Proline: 4 mg/mL  1 mL   40 μg/mL ITS + supplement  1 mL 6.25 μg/mLbovine insulin 6.25 μg/mL transferrin 6.25 μg/mL selenous acid 5.33μg/mL linoleic acid 1.25 mgmL BSA Sodium pyruvate  1 mL   1 mMPenicillin/Streptomycin  1 mL  100 U/mL penicillin  100 μg/mLstreptomycin

Cells were thawed using 37° C. water bath and quickly transferred toculture media in 15 mL tube, washing out the cryovial with 1 mL ofmedia. Cells were centrifuged for 5 mins at 400 g. Supernatant wasaspirated and cells resuspended in 5 mL complete culture media. Cellcount was performed and enough cells were harvested to form pellets ofbetween 2-2.5 ×10⁵ cells/pellet. 4 positive cultures and 2 negativecultures were set up for each sample. Cells were centrifuged again for 5mins at 400 g to remove culture media. Supernatant was aspirated andcells resuspended in 3 mL incomplete chondrogenic media (ICM). 3 mL cellsuspension was divided into 15 mL tubes (2 mL for positive pellets, 1 mLfor negative pellets). Tubes were centrifuged for 5 mins at 100 g. Cellsfor positive pellets were resuspended in 500 pL of complete chondrogenicmedia (CCM) for every pellet to be formed. CCM consists of ICM with 0.5μL of TGF-β per mL of ICM.

Cells for negative pellet were resuspended in 500 μL of ICM for everypellet to be formed. Cells were transferred to screw cap Eppendorf tubesand centrifuged for 5 mins at 100 g in a swing out rotor. Tube capsloosened to allow gas exchange and incubated in BSC at 37° C., 5% CO₂.Media was changed every second day by aspirating as much of the media aspossible without disturbing the pellet and replacing with either CCM orICM for positive pellets and negative pellets respectively. At day 21,cell pellets were harvested by aspirating off all the media and washingtwice in D-PBS. Pellets were allowed to air dry and 3 of the 4 positivepellets were used for GAG measurement and the other one was used forhistology. Pellets for GAG measurement were stored at −20° C. and pelletused for histology was fixed in 10% formalin for 1 hr and then stored inwater until ready to be processed.

Example 4—Chondrogenic Assay Preparation of DMMB Stock Solution

16 mg of DMMB was dissolved overnight in 5 mL of reagent grade 100%ethanol. 2.73 g NaCl and 3.04 g Glycine was added to 975 mL of deionisedwater. 0.69 mL of conc HCl (11.6M) was added to this solution and mixed.Dissolved DMMB was added to this solution. Container of DMMB was thenrinsed repeatedly with Dl water until all of DMMB solution wastransferred. pH was adjusted to 3.0 with 1 M HCl. Volume was adjusted to1 L with deionised water and solution was protected from light bywrapping in tinfoil.

Papain solution was prepared by dissolving 1 mg of papain (Sigma P4762)in 9.75 mL of warm diluted buffer. Diluted papain was prepared by adding250 μL of this solution to 10 mL of dilution buffer.

200 μL of papain solution was added to each pellet and allowed to digestovernight in 60° C. oven. Samples were then vortexed to disperse pellet.Standards were made up using chondroitin-6-sulphate (Sigma C4384) byadding 4 mg of chondroitin-6-sulphate to 10 mL of dilution buffer makinga 400 m/mL stock. This was then diluted to give a 40 μg/mL solution.Dilutions were made as follows from this 40 μg/mL solution as shown intable 3.

TABLE 3 Chondroitin sulphate Concentration solution Dilution GAG/well(40 μg/mL) Buffer (50 μL) 200 μL  0 μL   2 μg 180 μL  20 μL 1.8 μg 160μL  40 μL 1.6 μg 120 μL  80 μL 1.2 μg  80 μL 120 μL 0.8 μg  40 μL 160 μL0.4 μg  0 μL 200 μL   0 μg

50 μL of standards and samples were added in triplicate to each well ofa 96 well plate. 200 μL of DMMB stock solution was added to each welland incubated at room temperature (RT) for 5 mins. Plates were readusing an absorbance plate reader at 595 nm. Absorbance readings forstandards containing 0 μL GAG/well where used as a blank value andsubtracted from other absorbance readings.

Measurement of DNA Using PicoGreen

1×TE solution was prepared by diluting the 20×stock solution provided inthe Quant-iT Kit (Sigma P7589) 1 in 20 parts in distilled water. Adiluted PicoGreen solution was prepared by diluting DMSO to 1 in 200parts dH₂0. DNA stock (100 g/mL) was diluted in 1×TE 50-fold to give afinal concentration of 2 g/mL DNA standards were prepared as shown intable 4.

TABLE 4 DNA Working Final conc Stock 1 × TE DNA/mL 400 μL 0 2000 ng 200μL 200 μL 1000 ng 100 μL 300 μL  500 ng  40 μL 360 μL  200 ng  20 μL 380μL  100 ng  10 μL 390 μL  50 ng  4 μL 396 μL  10 ng  0 μL 400 μL   0 ng

Papain-digested samples (outlined above) were further diluted 25-fold in1×TE. 100 pL of standards and samples were added in triplicate to a96-well black plate. Plate must be black as reaction is affected bylight. 100 pL of PicoGreen solution was added to each standard andsample well and allowed to incubate for 2-3 mins. Plates read onfluorescent plate reader by first exciting plate at 485 nm and thenreading plate at 538 nm.

Example 5—Adipogenic Differentiation of SSC

Table 5 shows the composition of the adipogenic induction media.

TABLE 5 Volume (to make Reagent 100 mL) Final Concentration DMEM (HG)87.6 mL  1 μM Dexamethasone 1 mM  100 μL Insulin 1 mg/mL   1 mL  10μg/ml Indomethacin 100 mM  200 μL 200 μM 500 mM MIX  100 μL 500 μMPenicillin/Streptomycin   1 mL 100 U/mL penicillin 100 μg/mLstreptomycin FBS   10 mL 10%

Table 6 shows the composition of the adipogenic maintenance media.

TABLE 6 Volume (to make Reagent 100 mL) Final Concentration DMEM (HG) 88mL Insulin 1 mg/mL  1 mL  10 μg/ml Penicillin/Streptomycin  1 mL 100U/mL penicillin 100 μg/mL streptomycin FBS 10 mL 10%

Cells were thawed using 37° C. water bath and quickly transferred toculture media in 15 mL tube, washing out the cryovial with 1 mL ofmedia. Cells were centrifuged for 5 mins at 400 g. Supernatant wasaspirated and cells resuspended in 5 mL complete culture media. Cellcount was performed and enough cells were harvested to seed cells atconfluency (4 ×10⁴ cells/well) in a 24 well plate with flat bottom. 4test wells and 4 control wells were set up. Cells were seeded in 1 mL ofculture media in each well. Cells were incubated at 37° C., 5% CO₂ andafter 48 hrs cells were viewed to have adhered to the plastic andappeared confluent. To test wells, complete culture media was replacedwith 1 mL of adipogenic induction media and left for 3 days. Controlwells were replaced with complete culture media. After 3 days inadipogenic culture media, media in test wells was replaced with 1mL/well of maintenance media and left for between 1 and 3 days. This wasthen replaced with 1 mL/well of maintenance media. This process wasrepeated three times. After the final media change to maintenance media,cells were left in media for 5 to 7 days before harvesting.

Example 6—Adipogenic Assay Oil Red O Staining

A working solution of Oil Red O was prepared by mixing 6 parts of OilRed O stock solution with 4 parts of dH₂O. Solution was allowed to standfor 10 mins and then filtered through Whatman no. 1 Filter paper.

Media was aspirated and cells washed twice in D-PBS. Cells were thenfixed in 10% formalin for 1 hr at RT. Formalin was aspirated and platesrinsed in dH₂O. 500 pL of Oil Red O working solution was pipetted toeach well to cover layer of cells. Plate rotated slowly in FIG. 8 motionto spread Oil Red O over cells evenly and left for 5 mins. Stain wasaspirated and excess stain was removed by adding 2 mL/well of 60%Isopropanol. Plates were again swirled in FIG. 8 motion and Isopropanolaspirated. Plates rinsed with tap water until water ran off platesmoothly. Samples were then stored in dH₂0 until imaging.

Extraction of Stained Lipid

After imaging of samples, water was aspirated from wells. Oil Red O wasextracted by pipetting Isopropanol (2 ×500 pL) over the surface of thewells several times. Isopropanol and dye were then transferred to anEppendorf tube. Samples were centrifuged for 2 mins at 500 g to pelletand debris in samples. 200 μL of the extracted stain for each sample wasadded in triplicate to a 96 well plate. Staining was measured using aplate reader at 520 nm.

Example 7—Osteogenic Differentiation of SSC

Table 7 shows the composition of the osteogenic differentiation media.

TABLE 7 Volume (to make Reagent 100 mL) Final Concentration DMEM (LG)87.5 mL Dexamethasone 1 mM   10 μL 100 nM Ascorbic acid 2-P 10 mM   1 mL100 μM B glycerophosphate   1 mL  10 mM FBS   10 mL 10%Penicillin/Streptomycin   1 mL 100 U/mL penicillin 100 μg/mLstreptomycin

Cells were thawed using 37° C. water bath and quickly transferred toculture media in 15 mL tube, washing out the cryovial with 1 mL ofmedia. Cells were centrifuged for 5 mins at 400 g. Supernatant wasaspirated and cells resuspended in 5 mL complete culture media. Cellcount was performed and enough cells were harvested to seed cells atconfluency (4 ×10⁴ cells/well) in a 24 well plate with flat bottom. 4test wells and 4 control wells were set up. Cells were seeded in 1 mL ofculture media in each well. Cells were incubated at 37° C., 5% CO₂ andafter 48 hrs cells were viewed to have adhered to the plastic andappeared confluent. Media in test wells was replaced with osteogenicmedia and media in control wells was replaced with complete culturemedia. Media in all wells was changed twice weekly. Cells were harvestedbetween days 10 and 17.

Osteogenic Assay

1 of 4 test wells and control wells are used for Alizarin Red staining.The other 3 were used for calcium quantification.

Alizarin Red Staining

2% Alizarin Red S solution was prepared by dissolving 2 g Alizarin Red Sin 100 mL dH₂O. Solution was mixed and pH was adjusted to approximately4.1-4.3 using 1% ammonium hydroxide as pH is essential for stainingprocess. Media was aspirated from wells. Cells were washed twice inD-PBS to remove remaining media to insure no staining of media occurred.95% methanol was prepared by diluting 95 mL 100% methanol with 5 mLwater. Methanol was then stored in ice to low temperature. Cells werefixed in ice cold Methanol for 10 mins. Methanol was aspirated and cellswere rinsed in dH₂O. 500 pL of 2% Alizarin Red S was added to wells andleft for 5 mins, occasionally gently swirling the plate in FIG. 8motion. After 5 mins calcium staining was visible. Cells rinsed in dH₂Oand imaged using an Olympus CKx41.

Calcium Assay

0.5M HCl was prepared by diluting 4.3 mL 11.6M HCl in 95.7 mL water.Media was aspirated from wells and wells washed twice in D-PBS to removeany remaining media. 0.2 mL 0.5M HCl was added to each well and cellswere scraped from wells using a cell scraper and collected in labelledEppendorf tubes. Solution was left shaking overnight on cell shaker in adark cold room. Samples centrifuged briefly to pellet cell debris.Calcium assay was performed using a Stanbio Kit. A working solution of1:1 of binding reagent and working dye were prepared.

Table 8 shows the composition of calcium assay standards.

TABLE 8 Volume Concentration 10 mg/dl (μg/well) std/well 0 0 0.05 0.5 μL0.10   1 μL 0.2   2 μL 0.4   4 μL 0.6   6 μL 0.8   8 μL 1.0  10 μL

Standards and samples were plated in triplicate in a 96 well plate. 10μL of 0.5M HCl was added to each standard well. 10 pL of samples wereadded to each well. 200 μL of working solution was added to everystandard and sample well. Absorbance was read at 550-650 nm using aVictor3™ 1420.

SDC2 co-stained with Scal on the surface on CD45-ve murine BMMNC fromthe C57/B16 strain. Moreover, FACS sorting of SDC2+/Sca1+ MNC frommurine marrow reveals that SDC2 marks a self-renewing sub-population ofSSC that can form CFU-F at significantly increased frequencies comparedto plated pre-sorted MNC.

Example 8—SDC2⁺ Cells from Human Pluripotent Cells

We obtained populations of cells expressing SDC2 from human pluripotentcells, in this case ES cells (ES−), for comparison with cells derivedfrom bone marrow (BM−).

BM-SSCs and ES-SSCs (Millipore Human Mesenchymal Stem Cells (derivedfrom hES cells)) were plated at a density of 10⁵ cells per well of a6-well plate (Nunc) in complete media (BM-SSCs: α-MEM, 10% FBS; ES-SSCs:Millipore FibroGRO™ LS Complete Media Kit) and left to adhere overnight.Cells were harvested when they reached subconfluent levels (˜60%confluent), and confluent levels (100% confluent).

The results from flow cytometric analysis of “classical” SSC markersillustrated that BM-SSCs and ES-SSCs had similar expression of CD73. Theexpression of the marker CD105 remained the same for both confluent andsub-confluent cultures; CD105 expression appeared to decrease withincreasing confluency. The expression of SDC2 by BM- and ES-SSCsremained consistent in confluent and sub-confluent culture conditions;the percentage population BM-SSCs expressing SDC2 increases in confluentculture and is consistently high for ES-SSCs in both confluent andnonconfluent cultures. The RFI of SDC2 expression by ES-SSC is higher.

Hence, hES derived stromal stem cells expressed SDC2 and therefore cellpopulations enriched for SDC2 can be obtained direct from humanpluripotent cells including hES and hiPS cells.

Example 9—SDC2* Cells in Treatment of Ventilator Induced Lung Injury inRats Methods and Materials

All work was approved by the Animal Ethics Committee of the NationalUniversity of Ireland, Galway and conducted under license from theDepartment of Health, Ireland.

hSSC Isolation and Culture

Human SSCs (hSSCs) were isolated from adult volunteers as previouslydescribed. Following aspiration, the bone marrow was plated into tissueculture flasks. Adherent cells were grown until 80% confluent and thentrypsinized and culture expanded to passage 4, whereupon they were usedfor experiments. SSCs were characterized according to internationalguidelines. Fibroblasts, used as control cells, were obtained from astable cell line as previously described.

Series 1 [Ventilation Induced Lung Injury]

-   -   Adult male Sprague Dawley rats were anaesthetised, orotracheally        intubated and randomized to undergo injurious mechanical        ventilation.    -   The following ventilator settings were used: P_(Insp) 35 cmH₂O,        respiratory rate 18 min⁻¹, and PEEP 0 cmH₂0. When respiratory        static compliance had decreased by 50% the animals were allowed        to recover.    -   Following recovery, animals were randomized to intravenous        administration of: (i) vehicle (PBS, 300 μL); (ii) fibroblasts        (4 ×10⁶ cells); (iii) human SSCs (4 ×10⁶ cells) or (iv) cells of        the invention, referred to as human S2⁺SSCs (4 ×10⁶ cells); in a        four group design.    -   The extent of recovery following ALI and the inflammatory        response was assessed after 24 hours.

Series 2 [Low Stretch ‘Protective Ventilation]

-   -   Adult male Sprague Dawley rats were anaesthetised, orotracheally        intubated and randomized to low stretch mechanical ventilation.    -   The ‘low stretch’ protocol comprised of mechanical ventilation        for 90 minutes with the following settings: FiO₂ of 0.3,        respiratory rate 80.min⁻¹, tidal volume 6 ml·kg⁻¹ and positive        end-expiratory pressure of 2 cm H₂O    -   Following recovery, animals were randomized to intravenous        administration of: (i) vehicle (PBS, 300 μL); (ii) fibroblasts        (4 ×10⁶ cells); or (iii) intra-tracheal human SSCs (4 ×10⁶        cells); in a six group design.    -   The extent of recovery following ALI and the inflammatory        response was assessed after 24 hours.

Statistical Analysis

The distribution of all data was tested for normality usingKolmogorov-Smirnov tests. Data were analyzed by one-way ANOVA, followedby Student-Newman-Keuls, or by Kruskalis-Wallis followed by Mann-WhitneyU test with the Bonferroni correction for multiple comparisons, asappropriate. Underlying model assumptions were deemed appropriate on thebasis of suitable residual plots. A two-tailed p value of <0.05 wasconsidered significant.

Results

Efficacy of S²⁺SSCs in Enhancing Recovery from Ventilation Induced ALI

40 animals were entered into the experimental protocol, with 10allocated to each of the VILI groups. Four VILI animals, two allocatedto receive vehicle, and two allocated to receive fibroblasts, did notsurvive the injury protocol. All other animals survived the injuryprotocol and subsequent treatment allocation. 8 animals each wereentered into the vehicle control and fibroblast groups, while 10 animalseach received hSSCs and S2⁺SSCs.

Baseline Characteristics: There were no differences among the VILIgroups at baseline in terms of pre-injury variables, the duration ofinjurious ventilation or the extent of the lung injury produced (Table9).

TABLE 9 Baseline data regarding animals subjected to high stretchVentilation. High Stretch Ventilation Variable Vehicle Fibroblasts hSSCsS2⁺SSCs Number of animals 8 8 10 10 Animal Weight (g) 400 ± 26  392 ±51  410 ± 19  417 ± 18  Ventilation Time (mins) 76 ± 27 76 ± 16 77 ± 1978 ± 14 Lung compliance Pre-Injury (ml/mmHg 0.64 ± 0.09 0.66 ± 0.12 0.67± 0.13 0.66 ± 0.11 Lung compliance post-VILI 0.31 ± 0.02 0.32 ± 0.020.31 ± 0.03 0.32 ± 0.03 Note: Data are expressed as mean ± SD.

S2⁺SSCs restored lung function and structure following VILI: S2⁺SSCtherapy enhanced restoration of arterial oxygenation, as evidenced by areduced alveolar-arterial oxygen gradient compared to vehicle (p<0.05).Further functional recovery in lung physiology in response to S2⁺SSCtherapy was demonstrated by significant improvements (p<0.01) inrespiratory system static compliance in comparison to vehicle.

S2⁺SSCs improved lung microvascular permeability, as evidenced by adecrease in lung wet:dry weight ratios and a decrease in alveolar fluidprotein concentrations (Table 10). hSSCs enhanced recovery of lungstructure. S2⁺SSCs decreased alveolar thickening, as evidenced byreduced alveolar tissue volume fraction, and increased recovery ofairspace volume, as evidenced by increased alveolar air-space volumefraction (Table 10).

TABLE 10 Data regarding extent of resolution 24 hours following highstretch Ventilation. High Stretch Ventilation Variable VehicleFibroblasts hSSCs S2⁺SSCs Arterial O₂ tension (FiO₂ = 0.3; KPa) 13.4 ±2.8  12.7 ± 2.8  16.9 ± 2.9*  17.0 ± 1.7*  Arterial O₂ tension (FiO₂ =1.0; KPa) 32.1 ± 13.1 32.8 ± 16.0 65.3 ± 9.4*  56.2 ± 14.4* Lung StaticCompliance (ml/mmHg) 0.37 ± 0.04 0.34 ± 0.08 0.55 ± 0.14* 0.53 ± 0.08*Lung Wet Dry weight ratios 5.9 ± 0.8 5.4 ± 0.9 4.6 ± 0.2* 4.3 ± 0.7*Note: Data are expressed as mean ± SD. Final data is data collected uponcompletion of the experimental protocol. *Significantly differentvehicle and fibroblast groups.

S2⁺SSCs modulated inflammation following VILI: S2⁺SSCs decreased totalinflammatory cell counts in BAL (bronchoalveolar lavage) fluid andsubstantially attenuated (p<0.001) lung neutrophil accumulation. BothS2⁺SSCs and undifferentiated hSSCs were equally effective in modulatingthe inflammatory response following VILI (Table 11).

TABLE 11 Data regarding the inflammatory response 24 hours followinghigh stretch Ventilation. High Stretch Ventilation Variable VehicleFibroblasts hSSCs S2⁺SSCs BAL 2.91 ± 1.0  3.42 ± 0.86 1.30 ± 0.32* 1.50± 0.51* Cell Counts (×10 

 /ml) % BAL Neutrophils (%) 44.7 ± 12.2 56.7 ± 3.4  15.8 ± 8.5*  16.0 ±8.5*  BAL Neutrophil Counts (×10 

 /ml) 1.31 ± 0.60 1.92 ± 0.44 0.20 ± 0.10* 0.27 ± 0.22* BAL LymphocyteCounts (×10 

 /ml) 1.57 ± 1.02 0.94 ± 0.44 0.57 ± 0.14† 1.03 ± 0.67  Note: Data areexpressed as mean ± SD. Final data is data collected upon completion ofthe experimental protocol. *Significantly different vehicle andfibroblast groups. †Significantly different from vehicle Group

indicates data missing or illegible when filed

Effect on ‘non-injury’ parameters: There was no effect of S2⁺SSCs orundifferentiated hSSCs on arterial pH, PCO₂, bicarbonate, base excess,lactate or mean arterial pressure (data not shown).

Effect of S2⁺SSCs in Animals Following Low Stretch Ventilation

16 animals were entered into the experimental protocol, with 4 allocatedto each of the groups. All animals survived the injury protocol andsubsequent treatment allocation.

Baseline Characteristics: There were no differences among the protectiveventilation groups at baseline in terms of pre-injury variables, theduration of injurious ventilation or the extent of the lung injuryproduced (data not shown).

S2⁺SSCs did not affect lung function or structure: There was no effectof S2⁺SSC therapy on lung structure or function following protectiveventilation (Table 12).

TABLE 12 Data regarding extent of resolution 24 hours following lowstretch Ventilation. Low Stretch Ventilation Variable VehicleFibroblasts hSSCs S2⁺SSCs Arterial O₂ tension (FiO₂ = 0.3; KPa) 17.6 ±1.2  17.8 ± 0.8  17.8 ± 0.6  18.5 ± 0.7  Arterial O₂ tension (FiO₂ =1.0; KPa) 65.8 ± 1.7  69.2 ± 1.7  68.8 ± 3.3  64.3 ± 6.3  Lung StaticCompliance (ml/mmHg) 0.53 ± 0.03 0.59 ± 0.06 0.64 ± 0.02 0.61 ± 0.04Lung Wet:Dry weight ratios 4.3 ± 0.4 4.3 ± 0.5 4.2 ± 0.2 4.3 ± 0.6 Note:Data are expressed as mean ± SD. Final data is data collected uponcompletion of the experimental protocol.

S2⁺SSCs did not cause inflammation: There was no effect of S2+SSCstherapy on the inflammatory response in the lung structure followingprotective ventilation (Table 13).

TABLE 13 Data regarding the inflammatory response 24 hours following lowstretch Ventilation. Low Stretch Ventilation Variable VehicleFibroblasts hSSCs S2⁺SSCs BAL Cell Counts (×10 

 /ml) 1.24 ± 0.24 1.08 ± 0.13 1.01 ± 0.10 1.14 ± 0.32 % BAL Neutrophils(%) 11.3 ± 2.8  9.8 ± 2.1 20.8 ± 4.9  10.3 ± 2.0  BAL Neutrophil Counts(×10 

 /ml) 0.14 ± 0.06 0.10 ± 0.02 0.21 ± 0.04 0.11 ± 0.03 BAL LymphocyteCounts (×10 

 /ml) 0.64 ± 0.16 0.65 ± 0.38 0.65 ± 0.52 0.59 ± 0.31 Note: Data areexpressed as mean ± SD. Final data is data collected upon completion ofthe experimental protocol.

indicates data missing or illegible when filed

Effect on ‘non-injury’ parameters: There was no effect of S2⁺SSCs orundifferentiated hSSCs on arterial pH, PCO₂, bicarbonate, base excess,lactate or mean arterial pressure (Table 14).

TABLE 14 Data regarding ‘non-injury’ parameters 24 hours following lowstretch Ventilation Low Stretch Ventilation Variable Vehicle FibroblastshSSCs S2⁺SSCs Arterial pH 7.40 ± 0.04 7.39 ± 0.03 7.38 ± 0.03 7.40 ±0.04 Arterial PCO₂ (KPa) 5.4 ± 0.8 5.5 ± 0.2 5.0 ± 0.2 4.4 ± 0.3Arterial Bicarbonate (mMol/L) 20.5 ± 2.0  22.0 ± 1.5  20.9 ± 1.0  21.7 ±2.1  Base Excess 3.4 ± 1.5 3.3 ± 1.7 3.4 ± 2.0 2.8 ± 1.8 ArterialLactate (mMol/L) 3.1 ± 1.4 2.2 ± 0.6 2.1 ± 0.8 2.0 ± 1.2 Mean ArterialPressure (mmHg) 113.2 ± 2.7  101.0 ± 10.7  98.0 ± 13.7 99.5 ± 17.1 Note:Data are expressed as mean ± SD. Final data is data collected uponcompletion of the experimental protocol.

CONCLUSIONS

S2⁺SSCs of the invention restored lung function and structure followingVILI, as evidenced by a reduced alveolar-arterial oxygen gradient,significant improvements (p<0.01) in respiratory system staticcompliance, and improved lung microvascular permeability. Also, theyenhanced recovery of lung structure following VILI. The cells modulatedinflammation following VILI, decreasing total inflammatory cell countsin BAL fluid and substantially attenuating (p<0.001) lung neutrophilaccumulation. There was no effect of S2⁺SSC therapy on lung structure orfunction, or on the inflammatory response, following protectiveventilation. These findings suggest that the cells of the invention arewell tolerated in this model.

The invention thus provides methods of obtaining defined stromal stemcell populations and uses thereof.

1. A composition comprising: (i) a population of mammalian stromal stemcells, wherein 30% or more of the cells are positive for SDC2; and (ii)a cryopreservant, wherein the population of mammalian stromal stem cellsexhibits at least 10-fold more colony forming units per 10{circumflexover ( )}5 cells plated compared with native pre-sorted mononuclearcells.
 2. The composition of claim 1, wherein the cryopreservant isdimethylsulfoxide, human serum albumin, or hyaluronic acid.
 3. Thecomposition of claim 1, further comprising saline or collagen.
 4. Thecomposition of claim 1, wherein the cells are derived from bone marrow,umbilical cord, adipose tissue, skeletal muscle, endometrium, placenta,umbilical cord blood, or Wharton's jelly.
 5. The composition of claim 1,wherein 30% or more of the cells are pre-chondroblast cells.
 6. Thecomposition of claim 1, wherein 75% or more of the cells are positivefor SDC2.
 7. The composition of claim 1, wherein 75% or more of thecells are osteo-lineage precursor cells cells.
 8. The composition ofclaim 1, wherein less than 25% of the cells are pre-adipocyte cells. 9.The composition of claim 1, wherein the cells are human cells.
 10. Thecomposition of claim 1, wherein the cells are negative for CD45.
 11. Thecomposition of claim 1, wherein the composition is suitable forinjection.